Acidity-activatable upconversion afterglow luminescence cocktail nanoparticles for ultrasensitive in vivo imaging

Activatable afterglow luminescence nanoprobes enabling switched “off-on” signals in response to biomarkers have recently emerged to achieve reduced unspecific signals and improved imaging fidelity. However, such nanoprobes always use a biomarker-interrupted energy transfer to obtain an activatable signal, which necessitates a strict distance requisition between a donor and an acceptor moiety (<10 nm) and hence induces low efficiency and non-feasibility. Herein, we report organic upconversion afterglow luminescence cocktail nanoparticles (ALCNs) that instead utilize acidity-manipulated singlet oxygen (1O2) transfer between a donor and an acceptor moiety with enlarged distance and thus possess more efficiency and flexibility to achieve an activatable afterglow signal. After in vitro validation of acidity-activated afterglow luminescence, ALCNs achieve in vivo imaging of 4T1-xenograft subcutaneous tumors in female mice and orthotopic liver tumors in male mice with a high signal-to-noise ratio (SNR). As a representative targeting trial, Bio-ALCNs with biotin modification prove the enhanced targeting ability, sensitivity, and specificity for pulmonary metastasis and subcutaneous tumor imaging via systemic administration of nanoparticles in female mice, which also implies the potential broad utility of ALCNs for tumor imaging with diverse design flexibility. Therefore, this study provides an innovative and general approach for activatable afterglow imaging with better imaging performance than fluorescence imaging.


Materials characterization
Proton nuclear magnetic resonance ( 1 H NMR) spectra were measured by a Bruker Avance II 400 MHz NMR and Fourier transform infrared spectroscopy (FTIR) were measured by a Bruker INVENIO-R.Dynamic light scattering (DLS) was recorded on a Zetasizer Nano ZS (Nano ZS90, Malvern).TEM images were captured using an FEI Tecnai F20 transmission electron microscope operating at an acceleration voltage of 80 kV.UV-Vis and fluorescence spectra were recorded on a PerkinElmer Lambda 35 and an Edinburgh FLS980 spectrofluorometer, respectively.Fluorescence, afterglow luminescence images and afterglow luminescence spectra were recorded on an IVIS Spectrum imaging system (PerkinElmer, Inc.).A Fiber Optic Illuminator equipped with a 150 W halogen lamp was used as the light source for generating the afterglow luminescence.A fiber coupled with an 808 nm laser system was purchased from Changchun New Industries Optoelectronics Tech.Co. Ltd.

FTIR measurements
The freeze-dried C18-PEG2000-DA and C18-PEG2000-SA were thoroughly mixed with potassium bromide powder and ground into powder in a mortar.Then the powder mixture is pressed into transparent discs by a tablet press to allow infrared light to penetrate.Finally, the discs are placed in a Fourier transform infrared absorption spectrometer for analysis.Note that potassium bromide discs need to be measured to get a background signal before the measurement of samples.

Preparation of ASN, C-ASN, Bio-ASN, AIN, Bio-AIN, and R-AIN
A mixed THF solution (1 mL) containing MEHPPV (0.25 mg/mL) and C18-PEG2000-DA (2 mg/mL) was used to prepare ASN by rapidly injecting it into 1 × HEPES (9 mL, pH = 8.5) under continuous vigorous sonication.C-ASN were prepared in the same way as ASN.Bio-ASN nanoparticles were obtained by mixing C18-PEG-Biotin (0.25 mg/mL), MEHPPV (0.25 mg/mL), and C18-PEG2000-DA (2 mg/mL) in THF solution (1 mL), followed by the same procedures as above.For AIN, NCBS (0.05 mg/mL) and PS-b-PAA (0.4 mg/mL) were mixed in THF solution (1 mL) followed by the same sonication step.After sonication, THF was slowly removed with by a gentle nitrogen flow.The aqueous solution was filtered through a polyethersulfone (PES) syringe-driven filter (0.22 µm) (Millipore), and washed three times using 30 K centrifugal filter units (Millipore) under centrifugation at 1,300 g for 15 min.Bio-AIN was prepared by mixing C18-PEG-Biotin (0.05 mg/mL), NCBS (0.05 mg/mL), and PS-b-PAA (0.4 mg/mL) in THF solution (1 mL), followed by sonication and other same steps as above.R-AIN nanoparticles were obtained by mixing alkylated-rose bengal (a-RB, 0.01 mg/mL, Supplementary Fig. 15, 35), NCBS (0.05 mg/mL), and PS-b-PAA (0.4 mg/mL) in THF solution (1 mL), followed by the same procedures as above.The concentrations of nanoparticles were determined by UV-Vis absorption according to their absorption coefficients.The ASN and C-ASN solutions were finally concentrated to 0.5 mg/mL (based on the mass of MEHPPV) and AIN and R-AIN solutions were concentrated to 0.25 mg/mL (based on the mass of NCBS) by ultrafiltration and stored in dark at ~ 4 °C.

Afterglow and fluorescence luminescence imaging
Afterglow and fluorescence luminescence imaging were performed on an IVIS Spectrum imaging system in bioluminescence and fluorescence modes, respectively.For acquisition of afterglow luminescence images, samples were pre-illuminated by 808 nm laser at a power density of 1 W/cm 2 or white light at a power density of 0.1 W/cm 2 for 1 min unless otherwise noted.In vitro afterglow signals were collected for 10 s with an open filter.In vitro fluorescence signals were collected for 5 s with an excitation wavelength at 480 nm and emission wavelength at 580 nm.The afterglow spectra were collected for 5 s with specific emission filters.For in vivo experiments, the afterglow images were recorded for 30 s with an open filter, the fluorescence images were recorded for 5 s with an excitation wavelength at 480 nm and emission wavelength at 580 nm.The afterglow and fluorescence intensities were quantified by measuring the signal intensity of the region of interest (ROI) using Living Imaging 4.5 Software.

Subcellular localization study
HepG2 cells were seeded on a confocal dish (35 mm) with 1 × 10 4 cells per dish with 1 mL medium overnight.HepG2 cells were pretreated with ASN (40 μg/mL) or R-AIN (40 μg/mL) for 12 h, respectively.Next, the medium was removed and the cells were washed with PBS for three times.Then, the cells were then stained with commercial Lyso Green Tracker (10 μM), Mito Green Tracker (10 μM) and Hoechst 33342 (10 μM) for the nuclei.Fluorescence images were obtained on CLSM.For Lyso Green Tracker and Mito Green Tracker, an excitation wavelength was 488 nm with emission wavelengths at 510 nm ± 10 nm.For ASN, an excitation wavelength was 488 nm with emission wavelengths at 580 nm ± 10 nm.For R-AIN, an excitation wavelength was 559 nm with emission wavelengths at 580 nm ± 10 nm.

Cytotoxicity assay
HepG2 cancer cells were seeded in 96-well plates (8000 cells in 100 µL supplemented medium per well) and incubated for 24 h.Aqueous dispersions of ASN or C-ASN (final concentrations 1, 2, 5, 10, 20, and 30 µg/mL) and AIN (final concentrations 0.1, 0.2, 0.5, 1.0, 2.0, and 3.0 µg/mL) were introduced into the culture medium and incubated for 24 h.The cell culture medium was removed, and the fresh medium (100 µL per well) mixed with CCK-8 (5 mg/mL, 10 µL per well) was then added in the wells.After incubation for 4 h, the absorbance of CCK-8 at 450 nm was recorded by EnSpire multimode plate reader (PerkinElmer).Cell viability was calculated according to the ratio of absorbance of experimental well to that of the control cell well.
Blood circulation ASN (8 mg/kg) and AIN (0.8 mg/kg) were i.v.injected into mice, respectively.At different time points post-injection, blood samples were collected from the retinal vein for fluorescence assays.The blood samples were diluted by four-fold with 1 × HEPES buffer.The contents of ASN and AIN in the blood samples were determined through fluorescence by IVIS Spectrum imaging system (PerkinElmer, Inc.) with an excitation at 480 nm and emission at 580 nm (ASN), and an excitation at 720 nm and emission at 790 nm (AIN), respectively.

Biodistribution method
The mice were euthanized by CO2 asphyxiation 48 h after administration of ALCNs and C-ALCNs (n = 3).Major organs were collected and then placed onto black paper.All organs were pre-irradiated with an 808 nm laser (1 W/cm 2 ) or white light (0.1 W/cm 2 ) for 1 min, and the afterglow luminescence images were acquired for 10 s with an open filter using IVIS Spectrum imaging system.The fluorescence images were acquired for 5 s under an excitation wavelength at 480 nm and emission wavelength at 580 nm.The afterglow and fluorescence luminescence intensities for each individual organ were analyzed by the ROI analysis using the Living Image 4.5 Software.

Histology
After being fixed with 4% paraformaldehyde (PFA), the organs were dehydrated in ethanol solution and embedded in paraffin prior to 10 µm sectioning.Histology samples were stained by hematoxylin and eosin under standard protocols.Images were obtained on a confocal microscope (FV1200, Olympus).

Fig. 1 .
Schematic illustration for the pH-insensitive nanoprobe, C-ALCNs.Schematic illustration of the preparation of C-ASN through nanoprecipitation.No charge changes occurred at any pH and thus the 1 O2 transfer